Atletico Madrid, Inter Milan, AC Milan and Juventus have all dropped out of the Super League, after the six English clubs abandoned the planned competition, leaving the new competition essentially extinct before it even started.
Only Spanish clubs Real Madrid and Barcelona are now still officially involved.
The moves by Atletico, Inter, AC Milan and Juventus on Wednesday came a day after the six English Premier League clubs involved in the project made it unviable by giving up on the controversial breakaway competition.
Arsenal, Chelsea, Liverpool, Manchester United, Manchester City and Tottenham deserted plans for the largely-closed competition amid an escalating backlash from their supporters and warnings from the government that legislation could be introduced to thwart them.
The Super League project was overseen by Real Madrid president Florentino Perez, who promoted it as a way to "save soccer" and the clubs struggling financially amid the coronavirus pandemic.
Neither Madrid nor Barcelona commented after the rest of the clubs abandoned the project.
There was some internal pressure on the Catalan club, however, after outspoken captain Gerard Pique made his view clear.
"Football belongs to the fans. Today more than ever," he wrote on Twitter early Wednesday.
Barcelona coach Ronald Koeman avoided the subject but said he "agreed with" Pique's tweet.
Barcelona's presence in the new league was always conditioned on a vote by its general assembly.
The Spanish league planned to continue its campaign against the Super League with various actions and messages during matches on Wednesday and Thursday.
Defending league champions Madrid were playing at Cadiz later on Wednesday.
On Monday, UEFA threatened to ban players from the participating teams from playing in this year's European Championship and next year's World Cup.
But a Madrid court later issued a preliminary ruling stopping UEFA, FIFA and its members from acting against the creation of the new league.
AC Milan signalled it was leaving a few hours after Atletico and Inter Milan made their announcements.
"The voices and the concerns of fans around the world have clearly been expressed about the Super League, and AC Milan must be sensitive to the voice of those who love this wonderful sport," the Italian club said.
Juventus followed moments later, but didn't completely abandon plans for a future Super League.
Atletico said its decision was made after the board of directors met on Wednesday.
The Spanish club said it "decided to formally communicate the Super League and the rest of the founding clubs its decision not to formalise its participation in the project."
Atletico said the "circumstances" that allowed it to join the new league on Monday "no longer existed today."
Atletico fans had been expected to stage a protest before the home match against Huesca in the Spanish league on Thursday.
Widespread protests by fans in England played a big part in the decision by the Premier League clubs to leave the new competition.
"I knew the club would make the right decision and that's what happened," Atletico coach Diego Simeone said. "This is good for everyone."
The Super League was intended to be a 20-team competition with 15 founding members guaranteed a spot every season and five other teams rotating in and out.
The lack of relegation for the founding members raised concerns about the consequences for smaller clubs in the domestic leagues around the continent.